Clinical utility of novel mechanistic biomarkers of drug-induced liver injury

Abstracts / Toxicology Letters 221S (2013) S31–S56

Reference Laverty, H., Benson, C., Cartwright, E., Cross, M., Garland, C., Hammond, T., Holloway, C., McMahon, N., Milligan, J., Park, B., Pirmohamed, M., Pollard, C., Radford, J., Roome, N., Sager, P., Singh, S., Suter, T., Suter, W., Trafford, A., Volders, P., Wallis, R., Weaver, R., York, M., Valentin, J., 2011. How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines? Br J Pharmacol., 675–693, 163. Schultze, A.E., Walker, D.B., Turk, J.R., Tarrant, J.M., Brooks, M.B., Pettit, S.D., 2013. Current practices in preclinical drug development: Gaps in hemostasis testing to assess risk of thromboembolic injury. ToxicolPathol. 41, 445–453. Versteeg, H.H., Heemskerk, J.W.M., Levi, M., Reitsma, P.H., 2013. New fundamentals in hemostasis. Physiol Rev. 93, 327–358. Willeit, P., Zampetaki, A., Dudek, K., Kaudewitz, D., King, A., Kirkby, N.S., CrosbyNwaobi, R., Prokopi, M., Drozdov, I., Langley, S.R., Sivaprasad, S., Markus, H.S., Mitchell, J.A., Warner, T.D., Kiechl, S., Mayr, M., 2013. Circulating microRNAs as novel biomarkers for platelet activation. Circ Res. 112, 595–600.

http://dx.doi.org/10.1016/j.toxlet.2013.06.155

W10-2 Current status and future perspectives of renal safety biomarkers Mark Pinches, Amanda Benjamin, Stephen Furlong, David Brott AstraZeneca, Macclesfield, United Kingdom Renal toxicity is a significant cause of compound attrition during drug development. Drug induced renal toxicity arises though direct and indirect mechanisms which cause compromised renal function and structural injury. A wide number of biomarkers have been qualified for use preclinicallyto supplement plasma urea, plasma creatinine, urinalysis and histopathology. However, there remain several challenges for renal biomarkers including the need for greater validation in the context of drug safety, a better understanding of the impact of patient disease, and improved translatability between the preclinical species used in drug safety assessment and man and also between renal biomarker signals and glomerular filtration rate (GFR) estimations.Currently efforts are ongoing by a number of consortia and pharmaceutical companies to begin to address these challenges. Efforts are focused on extending the accepted context of use of these biomarkers to; additional species (including humans); additional toxicants; and defining patterns of biological variation. This presentation will describe the current state of the consortia activities; will present biological variation data from ahuman volunteer study; and in addition, will also present initial data from aunique detection kit that allows GFR measurements in conscious laboratory animal independent of blood and urine sampling. http://dx.doi.org/10.1016/j.toxlet.2013.06.156

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W10-3 Preclinical (epi)genomics – identifying safety biomarkers for the prediction of non-genotoxic carcinogenesis R. Terranova 1 , R. Luisier 1 , H. Lempiäinen 1 , A. Mueller 1 , M. Marcellin 1 , P. Couttet 1 , F. Bolognani 1 , V. Dubost 1 , E. Unterberger 2 , N. Scherbichler 1 , V. Vitry 1 , A. Del Espinola 1 , J. Thomson 3 , A. Braeuning 2 , J. Goodman 4 , O. Grenet 1 , D. Stiehl 1 , S-D. Chibout 1 , P. Moulin 1 , M. Schwarz 2 , R. Meehan 3 , E. van Nimwegen 5 , J. Moggs 1 1

Novartis Institutes for Biomedical Research, Basel, Switzerland, University of Tübingen, Department of Toxicology, Tübingen, Germany, 3 MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh, UK, 4 Center for Integrative Toxicology, Michigan State University, USA, 5 Biozentrum, University of Basel, Basel, Switzerland 2

Epigenetic mechanisms have an important role in human disease, and may also be involved in the pharmacodynamic and adverse effects associated with many drugs and toxicants. Nongenotoxic carcinogenesis (NGC) is a common drug-induced toxicity finding in rodent models for which no well-validated short-term assays exist. Here we have used genome-wide epigenomic and transcriptomic profiling, coupled to molecular histopathology, to investigate the temporal sequence of events and identify earlymechanisms and biomarkers of NGC in vivo in a well-characterized rodent model for liver tumor promotion. Exposure of mice to a tumor-promoting dose of phenobarbital (PB) led to transcriptional perturbations including the progressive increase in hepatic expression of non-coding RNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. Molecular histopathology and genetic analyses revealed the tissue-specificity and dependence to cancer-relevant pathways of the non-coding RNAs up-regulation. Computational modeling and the use of the Motif Activity Response Analysis (MARA) tool which models the expression dynamics in terms of predicted cis-regulatory sites led to the identification of transcription factor regulatory networks perturbed by PB treatment in liver. Finally, whole-genome methylome profiling identified precise perturbations of liver 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) patterns over the promoter regions of gene loci associated with cancer signalling pathways. Our data highlight novel potential early mechanisms and identify novel candidate early biomarkers for liver tumor promotion, providing new opportunities for assessing the carcinogenic potential of environmental cues including novel therapeutics. http://dx.doi.org/10.1016/j.toxlet.2013.06.157

W10-4 Clinical utility of novel mechanistic biomarkers of drug-induced liver injury Daniel J Antoine MRC Centre for Drug Safety Science, University of Liverpool, UK Drug-induced liver injury (DILI) represents a significant cause of patient morbidity, mortality and is a major contributor to attrition within drug development. Prediction of clinical DILI remains difficult, particularly in cases characterised by marked inter-individual variation. A lack of sensitivity, specificity and an indirect mechanistic basis of currently used biomarkers of hepatic injury remains

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Abstracts / Toxicology Letters 221S (2013) S31–S56

a factor for the delayed identification of DILI. Currently, ‘Hy’s law’ represents the regulatory endorsed model to predict serious DILI and the standard for novel DILI biomarkers to surpass. Pre-clinical biomarker identification and validation has been focused on molecular biomarkers such as cytokeratin-18, high mobility group box-1, glutamate dehydrogenase and micoRNA-122 from the perspective of acetaminophen toxicity (Antoine, 2009). These biomarkers hold translational application to inform the sensitive identification of DILI and its mechanistic basis in man (Starkey-Lewis, 2011. Antoine, 2012).A number of these biomarkers also provide enhanced prognostic information following acetaminophen overdose (Antoine, 2013]. However, significant challenges such as the characterization of inter- and intra-subject variability, as well as the impact of gender, age, and diurnal variation in healthy volunteersremain regarding these putative biomarkers as well as the utility in idiosyncratic DILI and the prediction of serious DILI from benign elevations in ALT activity. These challenges are the current focus of the Predictive Safety Testing Consortium (PSTC) and the IMI Safer and Faster Evidence based Translation (SAFE-T) consortium. The integrated use of these and qualification strategies will be discussed from a backdrop of imperfect current standards in the context of understanding fundamental hepatology.

and testing needs, the zebrafish embryo/larva model was explored as an alternative whole organism approach for DNT testing. First, a method was developed to investigate embryotoxic and teratogenic effects of compounds (Selderslaghs et al., 2012).Then 2 developmental stages of zebrafish were selected to monitor locomotor activity at non-teratogenic conditions as an endpoint for neurotoxicity: (i) spontaneous tail coiling of 1 day old embryos and (ii) swimming behaviour of hatched larvae (5-6 days). By means of video footage and tracking software, frequency and total duration of tail coiling in embryos (egg stage) and locomotor behaviourwith parameters for distance moved, mean velocity, turn angle,....in larvaewere studied. During an intra-laboratory validation exercise, these assays were evaluated for their ability to distinguish known developmental neurotoxicants (e.g. chlorpromazine, methylmercury,..) from negative compounds (Selderslaghs et al., 2013). The zebrafish results are compared to mammalian studies and discussed with respect to their predictive value for DNTassessment in higher vertebrates. It will be shown that zebrafish assays may fill a gap in the DNT test battery, especially when it concerns multi-target endpoints such as embryo development and behavior, which cannot be covered by cellular in vitro systems.

Reference Reference Antoine, D.J., Williams, D.P., Kipar, A., Jenkins, R.E., Regan, S.L., Sathish, J.G., Kitteringham, N.R., Park, B.K., 2009. High-mobility group box-1 protein and keratin-18, circulating serum proteins informative of acetaminopheninduced necrosis and apoptosis in vivo. Toxicol Sci. 112 (Dec (2)), 521– 531. Antoine, D.J., Jenkins, R.E., Dear, J.W., Williams, D.P., McGill, M.R., Sharpe, M.R., Craig, D.G., Simpson, K.J., Jaeschke, H., Park, B.K., 2012. Molecular forms of HMGB1 and keratin-18 as mechanistic biomarkers for mode of cell death and prognosis during clinical acetaminophen hepatotoxicity. J Hepatol. 56 (May (5)), 1070–1079. Antoine, D.J., Dear, J.W., Starkey-Lewis, P., Platt, V., Coyle, J., Masson, M., Thanacoody, R.H., Gray, A.J., Webb, D.J., Moggs, J.G., Bateman, D.N., Goldring, C.E., Park, B.K., 2013. Mechanistic biomarkers provide early and sensitive detection of acetaminophen-induced acute liver injury at first presentation to hospital. Hepatology (Feb), 6. Starkey Lewis, P.J., Dear, J., Platt, V., Simpson, K.J., Craig, D.G., Antoine, D.J., French, N.S., Dhaun, N., Webb, D.J., Costello, E.M., Neoptolemos, J.P., Moggs, J., Goldring, C.E., Park, B.K., 2011. Circulating microRNAs as potential markers of human druginduced liver injury. Hepatology 54 (Nov (5)), 1767–1776.

http://dx.doi.org/10.1016/j.toxlet.2013.06.158 Workshop 11: New approaches to unravel toxicities based on compound activity observed in zebrafish

W11-1 Locomotoractivity in zebrafish embryo and larva: alternative assays to evaluate the developmental neurotoxic potential of chemicals and drugs Hilda Witters 1 , Ingrid Selderslaghs 2 1 Flemish Institute for Technological Research (VITO), Environmental Risk and Health, Mol, Belgium, 2 University of Antwerp (UA), Biomedical Sciences, Wilrijk, Belgium

There is growing concern that chemical exposures contribute to the increasing incidence of neurodevelopmental diseases in children. However forchemicals currently in use,knowledge on their developmental neurotoxicity (DNT) is scarce while existing testguidelines (OECD, USEPA) to address regulatory demands do incur high costs and ethical drawbacks on animal use. To anticipate to 3 R

Selderslaghs, I.W., Blust, R., Witters, H.E., 2012. Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds. Reprod Toxicol. 33 (Apr 2), 142–154. Selderslaghs, I.W.T., Hooyberghs, J., Blust, R., Witters, H.E., 2013. Assessment of the developmental neurotoxicity of compounds by measuring locomotor activity in zebrafish embryos and larvae. Neurotoxicology and Teratology 37, 44–56.

http://dx.doi.org/10.1016/j.toxlet.2013.06.160

W11-2 Resolving the neuropharmacology of zebrafish sleep Jason Rihel University College London, United Kingdom The regulation and function of sleep remain among the fundamental mysteries of biology. Our work aims to unravel neural and molecular sleep regulators using zebrafish as a model system. Zebrafish larvae are optically transparent, easy to grow, and exhibit complex behaviors, including sleep, as early as 5 days post fertilization. They are also the only cost-effective vertebrate for genetic and chemical screens. We have developed a high-throughput assay to observe the effects of small molecules on zebrafish sleep/wake behavior.Larvae are placed into 96-well plates and observed by video camera continuously for several days.By screening thousands of small molecules, dissolved directly in the water, we uncovered both conserved and novel molecular pathways involved in sleep, including all the major neurotransmitter pathways, ether-a-go-go related gene (ERG) potassium channels, and verapamil-sensitive calcium channels.These results demonstrated the power of highthroughput behavioral profiling in zebrafish to discover and characterize psychotropic drugs and to dissect the pharmacology of complex behaviors.The screen also provided a large number of small molecule reagents to manipulate wild type and mutant zebrafish sleep. Our current efforts seek to identify the neuronal circuits modulated by small molecules using immediate early gene activation